THE PROTOSENSITIVITY OF CHICK-EMBRYO CELLS GROWING IN MEDIA CONTAINING CERTAIN CARCINOGENIC SUBSTANCES

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1 THE PROTOSENSITIVITY OF CHICK-EMBRYO CELLS GROWING IN MEDIA CONTAINING CERTAIN CARCINOGENIC SUBSTANCES MARGARET REED LEWIS (From the Drpcirtmrnt of Enibr,t/ology, Carnrgie Inntztutron of Wtcshiiigton, Bdtiinorr, Md.) When certain cancer-producing hydrocarbons were added to cultures of chick-embryo tissue, the cells developed photosensitivity to the electric light used for the study of cells in tissue cultures. The photodynamic action caused definite changes in the state of the cell protoplasm, which were often accompanied by inhibited cell division. This brought about a later occurrence of abnormalities of mitosis that duplicated many of the types of abnormal mitosis characteristic of malignant growths (Lewis arid Lewis, 1932 ; Lewis and Strong, 1934). METHOD Four samples of hydrocarbons were used in these experiments : two of 1 : 2 : 5 : 6-tlibenzanthracene (one obtained from Dr. A. Grollman and one from Dr. H. 3. Andervont), one of 1: 2-henzpyrene and one of methylcliolarithrene (both obtained from Dr. G. 0. Gey). The electric light was reflected from a W. Mazda 100 W. 115 V. round, frosted bulb covcred with :I gi-ecii shade, the edge of which was placed against the stage of the microscope in such a manner as to shield the eyes of the observer from The light, reflected by the mirror of the microscope, scrvecl to llluminatc the field under observation and the heat from the bulb kept the stage of the microscope at a temperature of 38" C. In this study of photodynamic action many types of cultures were used and various methods of introducing the hydrocarbons into the cultures were tried out. Among these were the following: hangingdrop cultures in which the tissue was explantcd into media (nutrient saline, chicken plasma, and mixtures of nutrient saline and serum) containing a suspension of hydrocarbon ; cultures in which the tissue 1f7as grown first in nutrient saline and the material sixspendcd in plasma added later ; cultures grown first in plasma and the hydrocarbon suspended in saline added later ; cultures in which the hydrocarbon was dissolved in chloroform, a drop of the solution evaporated on the coverslips, and the tissue explanted onto these films in hanging drops of chicken plasma. The suspension of hydrocarbon was prepared by grinding 0.5 to 1.0 mg. of the hydrocarbon together with one drop of chicken plasma or other nutrient medium in a miniature mortar. This was then diluted by adding chicken plasma and mixing until the desired concen- 305

2 306 MARGARET REED LEWIS t ration was reached. The amount of grinding did not seem important, as the resnlts were iiot dependciit upon the size of the particles making up the suspcnsioii. Care was t alteii to avoid undue evaporation during the preparation of the suspension. Tn some experiments 1 : 2 : 5 : 6- dihenzaritliraccne was first dissolved in lard, following Andc.rvo11t s (1934) directions for this procedure, and mixed into an emulsion with chicken plasma. The most satisfactory type of culture for these observations was thc hanging-drop culture iii which the tissue was allowed to grow for twenty-four hours in nutrient saline (Locke-Lewis solution), after which time the medium was replaced by a thin drop of a suspension of one of the hydrocarbons in chicken plasma. The culture was permitted to grow in this suspension from twenty-four to forty-eight hours bef orc exposure to the bright light. When a culture was selected for observation of the photodynamic action, one or two cells were first followecl through division under dull illumination. After this a field containing both resting cells and clividing cells was selected. An effort was made to include in the field at least oiie cell in the metaphase of division, for it had been found iii previous investigations that the reactions of cells to changes in their environment were readily discerned at this stage of mitosis. When such a field had been located, the oil-immersion, 2 mm. lens was adjusted and the condenser raised until the brightest light possible was secured. The changes in the cells brought about by this photodynamic action were then noted. BESULTS The presence in the cultures of 1 : 2 : 5 : 6-dibeiizanthraceiie, of 1 : 2- benzpyrene, or of metliylcliolaiithrciie in amounts up to 0.1 per cent did not interfere, to any marked extent, with the growth of the tissue. Tlic cells in these cultures and the cells in tlie control cultures were found to be similar in appearance and behavior except that in many of the dividiiig cells of the latter one or two chromosomes lagged at one of the poles of the spindle. While this lagging of the chromosomes did not occur in all of the cultures nor in all of the dividing cells of any one culture, it did take place often enough to make it seem characteristic of cells growing in the prcsencc of these hydrocarbons. The cells which were growing in cultures containing 1 : 2 : 5 : ti-dibenzantliracene dissolved in lard gave excellent results as far as photosensitivity was concerned, but observation was obscured and mitosis decreased due to tlie large number of fat globules stored in cells growing in such a fatty medium. In cultures containing small amounts of hydrocarbon and in the growths from explants which were not located near the hydrocarbo~l, the cells behaved normally even in the bright light. In cultures that had approximately 0.05 to 0.1 per cent hydrocarboll in their media, the cells remained undamaged and the process of mitosis continued in R normal manner while they were studied under dull

3 PHOTOSENSITIVITY OF CHICK-EMBRYO CELLS 307 illumination but, on exposure to bright light, the cells became changed and mitosis was inhibited within two to ten minutes. The first indication of change in the state of the protoplasm was an increase in Brownian movement of the fat globules in both the resting and the dividing cells. The mit ochoiidria remained extended as threads but became somewhat less uniform in width and more active in movement. Shortly after this the spindles of the cells in mitosis became shorter, while the mitochondria and other granules moved into the area previously occupied by the poles of the spindle. The decrease in spindle material continued until in many instances it entirely disappeared. When this happened, the chromosomes no longer remained in the equatorial plate but became spread out, forming a radiating figure resembling that of late prophase. Under these circumstances mitosis did not continue. In those cells in which the spindle did not entirely disappear the division, a1 though delayed, went on, and various abnormalities in the orderly- procedure of mitosis took place. In the resting cells the nucleolus became granular, the nucleus slifhtly coagulated and the nuclear wall became thicker and more refractive. Cells in cultures that had been growing in the presence of one of these hydrocarbons exhibited much greater photosensitivity than those in cultures to which the suspension of hydrocarbon had just been added, There was always considerable variability in effect on individual cultures of exposure to bright light. The amount of change that took place in the cells of a given culture depended upon the amount of hydrocarbon present, the thinness of the hanging-drop, the proximity of the cells to the particles of the material, the length of time the hydrocarbon had been in the culture, and the length of time the culture had been exposed to the bright light. When cultures that had reacted to the light were returned to the incubator before they were greatly damaged (within two to ten minutes), proliferation of the tissue started up again, and in the course of twenty-four hours the cells had largely recovered from their reaction to a short light exposure. Prolonged photodynamic action, in most instances, darnaged the cell beyond recovery. In order to demonstrate the changes in the state of the cytoplasm and the loss of tlie spindle, cultures that had and cultures that had not been exposed to light were fixed by means of an acid preservative and stained for permanent preparations (Lewis, R1. R., 1923). In the cultures fixed before exposure to bright light there were many metaphase figures containing mitotic spindles composed of bright coagulated fibrils. In those whose cells had reacted to the photodynamic action ihe spindles were either absent or short and only partly coagulated; few or none of the metaphase figures with the chromosomes arranged in a clear-cut equatorial band that are characteristic of all tissue growing in cultures were to he found. When a culture had been allowed to recover from the exposure to light before it was fixed, metaphase fig- ures with coagulated spindles were again plentiful in the stained prepar a t' ion.

4 308 iwar(:are?' REEI) LEWIS Most of the cultures that continued to proliferate for twenty-four to forty-eight hours after exposure to light exhibited many abnormal mitoses. These abnormalities were not of any one type, but coiisistcd of aberrant cliromosomes, lagging chromosomes, the addition of one or more extra chromosomes to one claughter cell with the consequent loss of those chromosomes from the other daughter cell, and a few tripolar spindles and multispiriclle cells. Cells with greatly increased or tetraploid numbers of chromosomes 011 one spindle were not observed, but otherwise the abnormalities were those characteristic of malignant t is su e. The photodynamic action brought about the same type of cellular response iii tissue growing in the presence of 1 : 2 : 5 : 6-dibeiizantliraeerie, of 1 : 2-bcnzpyrenc, aiid of methylcholanthrene. There was a difference iii the amount of each of these hydrocarbons that was nccessary to induce the photosensitivity as well as a difference in the rapidity with which the changes in the cells took place following exposure to light. On the whole, tlie amounts of tlie tliree hydrocarbons available and the degree of purity of the individual samples precluded any detailed comparison of the reaction to light exhibited by cells growing in the presence of the cancer-producing hydrocarbons studied. DISCUSSION The mariifestatioiis of the chaiiges in the state of the cytoplasm of the cells exposed to pliotodyiiamic action seemed to correspond in the first stages to those iiicluced by some of the influeiices (ether and ammonia, Roseiifeld, 1932, 1933 ; heat, Lewis, 1933, aiid distilled water, Lewis, 1934) that bring about a more fluid condition of tlie cytoplasm. In these cells tlie increase in fluidity coiitiiined in the dividing cells until the mitotic spinclle disappeared and tlie cells resembled in appearaiice those described in studies 011 rcvcrsible solatioii (Lewis, 1934). On blic other hand, in the restiiig cells, while the cytoplasm became more fluid, the condition of the nucleolus, the iiucleus, aiid tlie riuclcar wall resembled that described in cells following exposure to acid (Lewis, 1923, and Roseiifeld). The abnormal mitoses that occurred in cells that recovered from the photodyiiamic action corresponded to some extent with those fomid in tissue cultures that prolifcratcd following exposure to radium (Whitman, 1933). Many experiments were carried out iii mi effort to aiinlyxc the pheiiomeiia that take place in tlie cells growing in the presence of these hydrocarboiis upon exposure to light. Sirice it is known that these hydrocarbons are fluorescent, comparisoiis were made wi tli the photodynamic action of a iiumber of other fluorescelit substances. Also the action of varions degrees of lieat on cultures containing hydrocarbons was studied. On the whole, however, tlie investigations liave not determined wliether the photosensitivity of tlie cells is due to ohanges that have taken place in them while growing in the presence of the hydrocarbons or to chaiiges that liave occurred in the hydrocarbons while in the presence of grm7ing cells.

5 PHOTOSENSITIVITY OF CHICK-EMBRSO CELLS 309 LITERATURE ANDERVONT, H. B. : The productioii of diberizanthraeene tumors in pure strain niice, Public Health Reports 49 : 620, LEWIS, M. R.: Reversible gelation in living cells, Bull. Johns Hopkins Hosp. 34: 373, LEWIS, M. R.: Reversible changes in the nature of the mitotic spindle brought about in living cells by means of heat, hxh. f. exper. Zellforsch. 14: 464, LEWIS, M. R. : Reversible solation of the mitotic spindle of living chick eiribryo cells studied in vitro, Arch. f. exper. Zellforsch. 16: 159, LK:WIS, M. R., AND IIEWIS, W. H.: The malignant cells of Walker rat sarconi11 No. 338, Am. J. Cancer 16: 1153, LEWIS, M. R., AND LOCKWOOD, J. : The tetraploicl number of chromosomes in the malignttnt cell of the Walker rat sarcoma No. 1, Bull. Johns Hopkins Hosp. 44: 187, ~\VIS, &I. R., AXD SrrRom, L. c.: A study of spontaneous tumors of the mouse by the tissue culture method, Am. J. Cancer 20: 78, ~IOSENVELD, NI.: The action of ether on cells in mitosis, Arch. f. exper. Zellforsch. 12: 570, ROSENFELD, &I. : Experimental modification of mitosis by ammonia, Arch. f. exper. Zellforsch. 14: 1, ROYENFELD, M. : Unpublished observations. \VHITYAN, W. G. : Some observations on thc eflects of racliuni irradiation on tissue cdtures, Am. J. Cancer 17: 932, 1933.